초록 ▼

The invention provides isolated and recombinant phytase enzymes. In one aspect, the phytases are produced by modification of the wild type appA of E. coli. The enzyme can be produced from recombinant host cells. The phytases of the invention can be used to aid in the digestion of phytate where desi

The invention provides isolated and recombinant phytase enzymes. In one aspect, the phytases are produced by modification of the wild type appA of E. coli. The enzyme can be produced from recombinant host cells. The phytases of the invention can be used to aid in the digestion of phytate where desired. In particular, the phytases of the invention can be used in foodstuffs to improve the feeding value of phytate rich ingredients. The phytases of the invention can be thermotolerant and/or thermostable. Also provided are methods for obtaining a variant polynucleotide encoding a phytase and for obtaining a phytase with thermostability or thermotolerant at high or low temperatures.

대표청구항 ▼

What is claimed is: 1. An isolated or recombinant polypeptide comprising (a) a polypeptide having a phytase activity and an amino acid sequence having at least 98.5% sequence identity to a sequence as set forth in SEQ ID NO:2, or (b) a polypeptide having a phytase activity encoded by a nucleic acid

What is claimed is: 1. An isolated or recombinant polypeptide comprising (a) a polypeptide having a phytase activity and an amino acid sequence having at least 98.5% sequence identity to a sequence as set forth in SEQ ID NO:2, or (b) a polypeptide having a phytase activity encoded by a nucleic acid comprising a sequence having at least 98.5% sequence identity to SEQ ID NO:1. 2. The isolated or recombinant polypeptide of claim 1, wherein the phytase is thermotolerant. 3. The isolated or recombinant polypeptide of claim 1, wherein the phytase is thermostable. 4. The isolated or recombinant polypeptide of claim 1, wherein the nucleic acid encoding the polypeptide has at least 99% sequence identity to SEQ ID NO:1. 5. The isolated or recombinant polypeptide of claim 4, wherein the nucleic acid encoding the polypeptide has at least 99.5% sequence identity to SEQ ID NO:1. 6. The isolated or recombinant polypeptide of claim 1, wherein the amino acid sequence has a sequence as set forth in SEQ ID NO:2. 7. An isolated or recombinant polypeptide, wherein the polypeptide has a phytase activity, lacks a signal sequence, and comprises (a) a polypeptide having a phytase activity and an amino acid sequence having at least 98.5% sequence identity to a sequence as set forth in SEQ ID NO:2, or (b) a polypeptide having a phytase activity encoded by a nucleic acid comprising a sequence having at least 98.5% sequence identity to SEQ ID NO:1. 8. The isolated or recombinant polypeptide of claim 3, wherein the phytase activity is thermostable when heated to a temperature in the range from about 37째 C. to about 50째 C., about 50째 C. to about 70째 C. or about 70째 C. to about 90째 C. 9. The isolated or recombinant polypeptide of claim 8, wherein the phytase has a specific activity at about 37째 C. in the range from about 100 to about 1000 units per milligram of protein. 10. The isolated or recombinant polypeptide of claim 9, wherein the phytase has a specific activity from about 500 to about 750 units per milligram of protein. 11. The isolated or recombinant polypeptide of claim 3, wherein the phytase has a specific activity at 37째 C. in the range from about 500 to about 1200 units per milligram of protein. 12. The isolated or recombinant polypeptide of claim 11, wherein the phytase has a specific activity at 37째 C. in the range from about 750 to about 1000 units per milligram of protein. 13. The isolated or recombinant polypeptide of claim 2, wherein the phytase is thermotolerant after being heated to an elevated temperature in the range from about 37째 C. to about 90째 C. 14. The isolated or recombinant polypeptide of claim 13, wherein the phytase is thermotolerant after being heated to a temperature in the range from about 37째 C. to about 70째 C. 15. The isolated or recombinant polypeptide of claim 13, wherein the phytase retains at least half of its specific activity at 37째 C. after being heated to the elevated temperature. 16. The isolated or recombinant polypeptide of claim 13, wherein the phytase retains specific activity at 37째 C. in the range from about 500 to about 1200 units per milligram of protein after being heated to the elevated temperature. 17. The isolated or recombinant polypeptide of claim 1, wherein the polypeptide comprises at least one glycosylation site. 18. The isolated or recombinant polypeptide of claim 17, wherein the at least one glycosylation site is an N-linked glycosylation site. 19. The isolated or recombinant polypeptide of claim 1, wherein phytase is glycosylated after being expressed in P. pastoris or S. pombe. 20. The isolated or recombinant polypeptide of claim 1, wherein the polypeptide retains a phytase activity at a pH of about pH 5. 21. The isolated or recombinant polypeptide of claim 1, wherein the polypeptide retains a phytase activity at a pH of about pH 4. 5. 22. A protein preparation comprising a polypeptide as set forth in claim 1, wherein the protein preparation comprises a liquid, a solid or a gel. 23. A heterodimer or fusion protein comprising a polypeptide as set forth in claim 1 and a heterologous polypeptide. 24. The heterodimer or fusion protein of claim 23, wherein the heterologous polypeptide comprises an epitope. 25. The heterodimer or fusion protein of claim 23, wherein the heterologous polypeptide comprises a tag. 26. An immobilized polypeptide having a phytase activity, wherein the immobilized polypeptide comprises a polypeptide having a sequence as set forth in claim 1 or a heterodimer or fusion protein as set forth in claim 23. 27. The immobilized polypeptide of claim 26, wherein the phytase is immobilized on a cell, a metal, a resin, a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel, a plate, an array or a capillary tube. 28. An array comprising an immobilized polypeptide as set forth in claim 1 or claim 23. 29. A food supplement for an animal comprising a polypeptide as set forth in claim 1 having a phytase activity. 30. The food supplement of claim 29, wherein the polypeptide is glycosylated. 31. An enzyme delivery matrix comprising a polypeptide as set forth in claim 1 having a phytase activity. 32. The enzyme delivery matrix of claim 31, wherein the delivery matrix is in the form of a pellet, a pill, a tablet, a liquid, a gel, a capsule, a syrup, an emulsion, a lozenge, a dressing or a liposome. 33. The enzyme delivery matrix of claim 31, wherein the polypeptide is glycosylated. 34. The enzyme delivery matrix of claim 31, wherein the phytase is thermotolerant. 35. The enzyme delivery matrix of claim 31, wherein the phytase is thermostable. 36. An edible pellet comprising a granulate edible carrier and a polypeptide having phytase activity comprising a polypeptide as set forth in claim 1. 37. A feed composition comprising a polypeptide having a sequence as set forth in claim 1, and an edible carrier. 38. The feed composition of claim 37, wherein the phytase protein is a recombinant phytase protein. 39. The feed composition of claim 38, wherein the phytase protein is glycosylated. 40. The feed composition of claim 39 wherein the phytase protein is thermotolerant or thermostable. 41. The feed composition of claim 38, wherein the feed composition is manufactured in pellet, pill or tablet form. 42. The feed composition of claim 38, wherein the feed composition is produced using polymer coated additives. 43. The feed composition of claim 38, wherein the feed composition is manufactured in granulate form. 44. The feed composition of claim 38, wherein the feed composition is produced by spray drying. 45. An organic meal comprising a polypeptide as set forth in claim 1 having a phytase activity. 46. An isolated or recombinant polypeptide comprising (a) a polypeptide having an enzymatic activity and an amino acid sequence having at least 98.5% sequence identity to a sequence as set forth in SEQ ID NO:2, or (b) a polypeptide having an enzymatic activity and encoded by a nucleic acid comprising a sequence having at least 98.5% sequence identity to SEQ ID NO:1, wherein said enzymatic activity comprises catalyzing the hydrolysis of phytate (myo-inositol-hexaphosphate), or the reverse reaction. 47. The isolated or recombinant polypeptide of claim 46, wherein the sequence identity to the polypeptide of SEQ ID NO:2 is at least 99%. 48. The isolated or recombinant polypeptide of claim 47, wherein the sequence identity to the polypeptide of SEQ ID NO:2 is at least 99.5%. 49. The enzyme delivery matrix of claim 31, wherein the enzyme delivery matrix is an edible enzyme delivery matrix. 50. The isolated or recombinant polypeptide of claim 46, wherein the sequence identity to the nucleic acid of SEQ ID NO:1 is at least 99%. 51. The isolated or recombinant polypeptide of claim 50, wherein the sequence identity to the nucleic acid of SEQ ID NO:1 is at least 99.5%. 52. The organic meal of claim 45, wherein the organic meal comprises corn, soybean or a corn and soybean meal mix. 53. The isolated or recombinant polypeptide of claim 1, wherein the sequence identity to the polypeptide of SEQ ID NO:2 is at least 99%. 54. The isolated or recombinant polypeptide of claim 53, wherein the sequence identity to the polypeptide of SEQ ID NO:2 is at least 99.5%.